The present invention relates to a method of quantitatively or semi-quantitatively determining an analyte in a sample, especially a high concentration analyte.
For qualitative and quantitative determination of an analyte in a sample, a so-called sandwich assay is often used, wherein two receptors directed against different epitopes of the analyte are incubated with a sample containing the analyte, one of the receptors being detectable, e.g. through a label conjugated thereto. In a heterogeneous assay format, the second receptor is immobilized (e.g. coupled) to a solid phase or provided with a binder component, such as biotin, capable of binding to the solid phase, such as an avidin- or streptavidin-coated solid phase.
Especially in case the analyte is present in the sample in a high concentration, it is customary to dilute the sample before performing the assay to avoid the use of large and often costly amounts of immobilized receptor and labelled receptor, respectively, or to avoid technical difficulties where large amounts of receptors cannot be used. Such dilution is not only laborious but also introduces an additional source of error into the assay.
There is therefore a need of an assay procedure that avoids the necessity of dilution.
It is an object of the present invention to provide a method of performing a heterogeneous sandwich assay which permits the determination of even a high concentration analyte in a sample without the need to dilute the sample.
It is another object of the invention to provide a method of performing a heterogeneous sandwich assay which reduces the amounts of capturing and detection reagents used.
It is still another object of the invention to provide test kits for carrying out the method.
In one aspect of the present invention there is therefore provided a method of determining an analyte in a sample, especially a high concentration analyte found in concentrations greater than 1 nmole/liter, comprising the steps of:
a) contacting the sample with a specified amount of a receptor which binds specifically to the analyte to form an analyte/receptor complex, said specified amount of receptor being in excess of that required to bind all analyte in the sample,
b) isolating on a solid phase, preferably a matrix such as a membrane strip, a specified fraction of the amount of receptor contacted with the analyte, including analyte/receptor complex and unreacted receptor,
c) detecting the amount of analyte/receptor complex in said isolated specified fraction, and
d) from the detected amount analyte/receptor complex, determining the concentration of analyte in the sample.
In another aspect of the present invention there is provided a test kit for determining an analyte in a sample, comprising (i) a specified amount of a receptor substance having a first part which binds specifically to the analyte, and (ii) a solid phase member having immobilized thereon a ligand which binds specifically to a second part of the receptor, the amount of said ligand on the solid phase member being less than said specified amount of the receptor substance.
In still another aspect of the present invention there is provided a test kit for determining an analyte in a sample, comprising (i) a specified amount of a receptor substance having a first part which binds specifically to the analyte, only a specified fraction of the amount of receptor substance having a second part capable of binding to a specific ligand, and (ii) a solid phase member having said specific ligand immobilized thereon.
In yet another aspect of the present invention there is provided a test kit for determining an analyte in a sample, comprising (i) a first specified amount of a receptor substance, and (ii) a solid phase member having immobilized thereon a second specified amount of the receptor substance.
While it is preferred to use the method and test kit for quantitative determination of analytes of interest, they may also be used for semi-quantitative and qualitative determinations.